Genome Sequence Analysis of First Ross River Virus Isolate from Papua New Guinea Indicates Long-Term, Local Evolution

viruses

Communication Genome Sequence Analysis of First Ross River Virus Isolate from Papua New Guinea Indicates Long-Term, Local Evolution

Alice Michie 1, John S. Mackenzie 2,3,4 , David W. Smith 2 and Allison Imrie 1,*

1 School of Biomedical Sciences, University of Western Australia, Nedlands, WA 6009, Australia; [email protected] 2 PathWest Laboratory Medicine Western Australia, Nedlands, WA 6009, Australia; [email protected] (J.S.M.); [email protected] (D.W.S.) 3 School of Chemistry and Molecular Biosciences, University of Queensland, St. Lucia, QLD 4072, Australia 4 Faculty of Health Sciences, Curtin University, Bentley, WA 6102, Australia * Correspondence: [email protected]

Abstract: Ross River virus (RRV) is the most medically significant mosquito-borne virus of Australia, in terms of human morbidity. RRV cases, characterised by febrile illness and potentially persistent arthralgia, have been reported from all Australian states and territories. RRV was the cause of a large- scale epidemic of multiple Pacific Island countries and territories (PICTs) from 1979 to 1980, involving at least 50,000 cases. Historical evidence of RRV seropositivity beyond Australia, in populations of Papua New Guinea (PNG), Indonesia and the Solomon Islands, has been documented. We describe the genomic characterisation and timescale analysis of the first isolate of RRV to be sampled from PNG to date. Our analysis indicates that RRV has evolved locally within PNG, independent of Australian lineages, over an approximate 40 year period. The mean time to most recent common Citation: Michie, A.; Mackenzie, J.S.; ancestor (tMRCA) of the unique PNG clade coincides with the initiation of the PICTs epidemic in Smith, D.W.; Imrie, A. Genome mid-1979. This may indicate that an ancestral variant of the PNG clade was seeded into the region Sequence Analysis of First Ross River Virus Isolate from Papua New Guinea during the epidemic, a period of high RRV transmission. Further epidemiological and molecular- Indicates Long-Term, Local Evolution. based surveillance is required in PNG to better understand the molecular epidemiology of RRV in Viruses 2021, 13, 482. https:// the general Australasian region. doi.org/10.3390/v13030482 Keywords: arbovirus; mosquito-borne disease; alphavirus; Papua New Guinea; Australia Academic Editors: Bas B. Oude Munnink, Marietjie Venter and Luisa Barzon 1. Introduction Received: 15 February 2021 In Australia, the most common mosquito-borne viral infection is caused by Ross Accepted: 12 March 2021 River virus (RRV), an alphavirus of the family Togaviridae [1]. Disease associated with RRV Published: 15 March 2021 infection is characterised by potentially persistent and debilitating fatigue, myalgia, and arthralgia and/or arthritis. RRV cases have been reported from all Australian states and Publisher’s Note: MDPI stays neutral territories since it was first isolated from Aedes vigilax mosquitoes sampled in far-north with regard to jurisdictional claims in Queensland in 1959 [2]. published maps and institutional affil- iations. RRV is currently considered to be both a mosquito vector species and amplifying host generalist, with macropods (such as wallabies and kangaroos) considered the major reservoir host for RRV maintenance and transmission [3]. Humans have been implicated as short-term amplifying hosts during disease epidemics including the large-scale, virgin-soil epidemic that occurred across multiple Pacific Island countries and territories (PICTs) from Copyright: © 2021 by the authors. 1979 to 1980 [4–6]. Cases were reported from Fiji, American Samoa, the Cook Islands, Licensee MDPI, Basel, Switzerland. New Caledonia and Wallis and Futuna, with disease suspected in northern Tonga [7]. This article is an open access article Samoa did not report cases during this time, but during an April 1980 investigation of a distributed under the terms and conditions of the Creative Commons dengue outbreak, high titres of anti-RRV haemagglutination inhibition (HI) antibody were Attribution (CC BY) license (https:// detected. RRV activity likely occurred in Samoa during the 1979–1980 outbreak but went creativecommons.org/licenses/by/ undetected [7]. Viraemic travelers are the suspected mode of rapid spread between these 4.0/). adjacent island nations [8].

Viruses 2021, 13, 482. https://doi.org/10.3390/v13030482 https://www.mdpi.com/journal/viruses Viruses 2021, 13, 482 2 of 8

RRV transmission seemingly ceased in the region in mid-1980, presumably due to the lack of efficient reservoir hosts in the area [7,8]. A viraemic traveler from eastern Australia has been postulated as the source of the outbreak, which began in Fiji in April 1979, based on sequence similarity of epidemic viruses to the ‘Eastern-Australian’ lineage of RRV [9]. The geographical basis of RRV lineage nomenclature was not supported in our recent, more comprehensive genome-scale RRV phylogenetic analysis, where we showed that virus isolates that clustered as distinct lineages were geographically dispersed and sampled Australia wide [10]. The exact origins of the PICTs epidemic is now less certain, given viruses similar to those sampled during the epidemic, were sampled across Australia between 1982 and 1988 [10]. Four distinct genotypes of RRV (G1–4) have been characterised, with G4 in contemporary circulation across Australia [10]. RRV seropositivity has been described in the adult and general population of many regions of Papua New Guinea (PNG), sampled from 1960 to 1969, as well as regions of In- donesia and the Solomon Islands [11]. A later study found a 59% RRV seroprevalence in the Southern Highlands of PNG in 1991, and endemicity was suggested by increasing antibody prevalence with age [12]. RRV as a cause of arthritis in PNG has been confirmed [13,14]. RRV has been isolated from several mosquito species within Australia that are also present in PNG including Ae. vigilax, Ae. notoscriptus and Mansonia uniformis [15,16]. The exact prevalence, distribution, and genetic diversity of RRV in PNG is currently unknown, due to the lack of comprehensive surveillance in the region [17]. RRV cases were not reported from PNG during the PICTs epidemic. The first and only isolation of RRV from PNG to date was made in 1997 from a pool of Anopheles farauti mosquitoes collected at Wandoo village in the Bensbach region of the Western Province [16]. In this study, the genome sequence of this PNG isolate was derived and analysed with available RRV genome sequences to better understand the molecular epidemiology of RRV in the greater Australasian region.

2. Materials and Methods 2.1. Virus Isolation, RNA Extraction and Next Generation Sequencing The virus isolate, PNG3075, was isolated from a pool of An. farauti mosquitoes trapped in 1997 from the Bensbach locality of the Western Province of PNG [16]. The isolate was identified as RRV using a tissue culture-based fixed cell enzyme immunoassay involving specific, identifying monoclonal antibodies on fixed C6/36 monolayers (Ae. albopictus larvae, ATCC® CRL-1660™)[16,18]. The isolate was not investigated on a molecular level at the time of its initial characterisation. PNG3075 was amplified in the present study through a single passage on Vero cells (African Green Monkey kidney epithelium, ATCC® CCL-81™) maintained in Dulbecco’s Modified Eagle Media (Gibco DMEM, Thermo Fisher Scientific, Waltham, Massachusetts, USA) supplemented (by volume) with 5% heat-inactivated fetal bovine serum (FBS), 1% L- glutamine (Life Technologies, Carlsbad, California, USA) and 1% penicillin/streptomycin (Life Technologies). The Roche High Pure RNA extraction kit (Roche, Basal, Switzer- land) was utilised as per the manufacturers protocol from viral supernatant, concentrated through ultra-centrifugation in Millipore Amicon Ultra-15 centrifugal units (Merck Milli- pore, Darmstadt, Germany). The TruSeq stranded mRNA kit (Illumina, San Diego, California, USA) was used for library preparation. Superscript III reverse transcriptase (Invitrogen, Waltham, Mas- sachusetts, USA) was used for cDNA synthesis. Libraries were validated using the Agilent 1000 DNA kit (Agilent Technologies, Santa Clara, California, USA) and subsequently nor- malized and pooled. Sequence data were generated on the MiSeq platform with the MiSeq Micro v2 kit (Illumina). Paired-end reads of 150 bp were demultiplexed and assessed for quality within the FastQC v0.11 program [19], and subject to de novo genome sequence generation within CLC Genomics Workbench v7.5 (QIAGEN, Hilden, Germany) to derive a full-coding contiguous sequence, 11,837 nucleotides in length. A total of 567,724 reads Viruses 2021, 13, 482 3 of 8

were generated for the PNG3075 sample, of which 86.89% (493,294 reads) were used to generate the complete coding PNG3075 sequence.

2.2. Phylogenetic Analysis The derived genome was aligned with 104 publicly available RRV genome sequences using MAFFT 7.338 within Geneious 11.1.4 [20]. Most available sequence data were derived in our recent RRV phylogenetic investigation, with isolates primarily sampled in Western Australia [10]. For phylogenetic analysis, sequences were manually trimmed to remove 50 and 30 untranslated regions (UTRs) and then realigned. A maximum likelihood phylogeny was reconstructed using RAxML 8.2.11 with 1000 bootstrap replicates within Geneious 11.1.4, under a GTR+G+I nucleotide substitution model—the best-suited model according to JModelTest 2 analysis [21,22]. All phylogenies were visualized and edited within FigTree v1.4.4 [23].

2.3. Temporal Analysis The timescale of divergence of major RRV clades was estimated using Bayesian Markov chain Monte Carlo (MCMC) analysis within the BEAST package (v1.8) [24]. The dataset demonstrated considerable temporal signal (R2, 0.921; Correlation Coefﬁcient, 0.96) in root-to-tip regression analysis using TempEst v1.5 [25]. An uncorrelated lognormal (UCLN) clock was assumed under a GTR+G+I nucleotide substitution model, and a constant coalescent population with default priors. Three independent chains were run, assessed for convergence with log and tree ﬁles subsequently combined using LogCombiner. A maximum clade credibility tree (MCC) was reconstructed with the 105-taxon dataset, with 10% burn-in, and visualized using FigTree v1.4.

2.4. Accession Number The sequence derived in this study was deposited to the NCBI database and assigned the accession number MW238766.

3. Results Genome sequence analysis of isolate PNG3075 confirmed it as RRV, as initially identified through monoclonal antibody analysis when first isolated. A maximum likelihood (ML) phylogeny of a 105-taxon alignment of RRV complete coding sequences was re-constructed (Figure S1). PNG3075 constituted a unique clade, distinct from all RRV isolates sampled in Australia over a 59 year period (1959–2018, Figure1). This is consistent, when a phylogeny is constructed from an alignment of a larger dataset of geographically defined partial genome sequences, sampled over the same timeframe (Figure S2). The unique clade is distinct from the genotype 4 (G4) viruses that were in wide Australian circulation at the time of the 1997 sampling of PNG3075. The long-terminal branch of the clade is suggestive of long-term evolution, independent of Australian lineages. Rather than a recent, or continuous exchange of virus between Australia and PNG, this clade was likely introduced into PNG in an historical event with subsequent local evolution. PNG3075 was most similar to isolates K1008 (Kununurra, 1986–genotype 3, G3) and DC7053 (Mandurah, 2005–G4), with 98.9% and 98.6% pairwise sequence identity, respectively. Viruses 2021, 13, x FOR PEER REVIEW 4 of 8 Viruses 2021, 13, 482 4 of 8

Figure 1. Maximum clade credibility (MCC) phylogeny of the 105-taxon Ross River virus (RRV) dataset. Clades are coloured Figure 1. Maximum clade credibility (MCC) phylogeny of the 105-taxon Ross River virus (RRV) dataset. Clades are col- foroured the genotypefor the genotype or sub-lineage or sub as-lineage characterised as characterised by the maximum by the maximum likelihood likelihood phylogeny phylogeny of Figure S1 of (seeFigure key S1 in lower(see key left). in Posteriorlower left). probability Posteriorvalues probability of > 0.70 values are of presented > 0.70 are above presented nodes, above as indicated nodes, byas indicated asterisks (*).by Theasterisks table (*) in. theThe upper-right table in the cornerupper presents-right corner the mean presents time the to most mean recent time commonto most ancestorrecent common (tMRCA) ancestor with error (tMRCA) reported with as the error 95% reported highest probabilityas the 95% densityhighest (95%probability HPD) fordensity each major(95% HPD) genetic for group each major of RRV. genetic The PNG group clade of RRV. has been The highlightedPNG clade has within been the highlighted table. within the table. A 36-nucleotide insertion within the nsP3 gene, that is characteristic of all RRV G3 and G4A isolates,36-nucleotide was also insertion present within in the the PNG3075 nsP3 gene, genome that [ 10is]. characteristic The earliest sampledof all RR virusV G3 containingand G4 isolates, this insertion, was also present is F9073—the in the PNG3075 ﬁrst isolate genome sampled [10] during. The earliest the PICTs sampled epidemic, virus fromcontaining Fiji in Aprilthis insertion, 1979. is F9073—the first isolate sampled during the PICTs epidemic, fromSeveral Fiji in April RRV isolates1979. have been observed to contain nucleotide insertions or deletions (indels)Several within RRV the isolates nsP3, have of various been observed sizes, mostly to conta affectingin nucleotide one of insertions the four or conserved deletions proline(indels) motifswithin ofthe the nsP3, nsP3 of hypervariablevarious sizes, mostly domain affecting [10]. No one such of the indels four were conserved observed pro- withinline motifs PNG3075. of the nsP3 hypervariable domain [10]. No such indels were observed within PNG3075.To determine the timescale of divergence of the PNG clade from Australian lineages of RRV,To thedetermine time to mostthe timescale recent common of divergence ancestor of (tMRCA)the PNG clade was estimated from Australian using Bayesian lineages methods,of RRV, the and time a maximum to most recent clade common credibility ancestor (MCC) (tMRCA) phylogeny was re-constructed estimated using (Figure Bayesian1). Anmethods, uncorrelated and a maximum lognormal, clade relaxed credibility molecular (MCC) clock phylogeny was assumed re-constructed based on results (Figure of 1). a generalAn uncorrelated sampling lognormal stepping-stone , relaxed analysis, molecular when comparedclock was withassumed a strict based clock on assumption. results of a Thegeneral GTR sampling + G + I nucleotide stepping-stone substitution analysis, model when was compared employed, with with a strict a constant clock assumption. coalescent tree prior. The GTR + G + I nucleotide substitution model was employed, with a constant coalescent The median root age of the entire 105-taxon dataset was estimated as July 1924 (mean: tree prior. 1924.5, 95% highest probability density (HPD) 1904.7–1941.1) under a relaxed clock. The The median root age of the entire 105-taxon dataset was estimated as July 1924 (mean: estimated mean nucleotide substitution rate was 3.06 × 10−4 (95% HPD 2.62–3.54 × 10−4), 1924.5, 95% highest probability density (HPD) 1904.7–1941.1) under a relaxed clock. The consistent with previous estimates of alphavirus evolutionary rates [10,26,27]. Each major estimated mean nucleotide substitution rate was 3.06 × 10−4 (95% HPD 2.62–3.54 × 10−4), genotype of RRV, as well as the newly described PNG clade, emerged in approximately consistent with previous estimates of alphavirus evolutionary rates [10,26,27]. Each major decade-long intervals, consistent with our previous genome-scale investigation of RRV [10].

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Viruses 2021, 13, 482 genotype of RRV, as well as the newly described PNG clade, emerged in approximately5 of 8 decade-long intervals, consistent with our previous genome-scale investigation of RRV [10]. ThThee mean tMRCAtMRCA ofof the the PNG PNG clade clade was was estimated estimated as Julyas July 1979 1979 (mean (mean 1979.5, 1979.5, 95% HPD95% HPD1972.6–1984.9), 1972.6–1984.9), corresponding corresponding to the to initiation the initiation of the of PICTs the PICTs epidemic epidemic in Fiji in (April Fiji (April 1979) 1979)and subsequentand subsequent spread spread to neighbouring to neighbouring islands islands in in the the region region (Figure (Figure2). 2). The The mean mean tMRCAtMRCA estimate estimate of of isolates isolates sampled sampled close close to to the the conclusion conclusion of of the the PICTs PICTs epidemic, from thethe Cook Cook Islands, Islands, is is comparable comparable to to that that of of the the PNG PNG clade clade estimates estimates (mean(mean 1979.9, 1979.9, 95% 95% HPDHPD 1979.4 1979.4–1980.0),–1980.0), though though with a narrower 95% HPD interval.

Figure 2. 2. MapMap of ofthe the Pacific Pacific region region and and the thelocation location of Pacific of Pacific Island Island Countries Countries and Territories and Territories (PICTs) (PICTs) involved, involved, or neigh- or bouring,neighbouring, the explosive the explosive Ross River Ross virus River (RRV) virus (RRV)epidemic epidemic that occurred that occurred between between 1979 and 1979 1980. and Orange 1980. Orange dots highlight dots highlight PICTs where confirmed RRV activity with virus isolations was documented, while blue dots highlight locations where RRV PICTs where confirmed RRV activity with virus isolations was documented, while blue dots highlight locations where RRV activity was suspected during the outbreak. The proximity of Papua New Guinea to these areas of activity is of note. The activity was suspected during the outbreak. The proximity of Papua New Guinea to these areas of activity is of note. The scale bar is shown in the lower-centre of the map. scale bar is shown in the lower-centre of the map. Our temporal estimates suggest that an ancestral variant of the PNG clade may have Our temporal estimates suggest that an ancestral variant of the PNG clade may have been seeded into PNG during the PICTs epidemic, a period of high RRV transmission in been seeded into PNG during the PICTs epidemic, a period of high RRV transmission the general Pacific region. It is not known whether virus was seeded to PNG directly from in the general Pacific region. It is not known whether virus was seeded to PNG directly thefrom PICTs the PICTs at the at height the height of the of outbreak, the outbreak, or from or from Australia Australia immediately immediately before, before, after, after, or concurrentlyor concurrently with with the theepidemic. epidemic. RRV RRV activity activity in PNG in PNG prior prior to the to PICTs the PICTs epidemic epidemic is evi- is dentevident from from several several historical historical seroprevalence seroprevalence studies, studies, but but molecular molecular inform informationation from from this this periodperiod is is lacking. lacking. Seroprevalence Seroprevalence data data from from 1960 1960 to 19 1969,69, encompassing multiple regions inin PNG, PNG, predate predate the the temporal temporal range range of of this this clade, suggesting suggesting that that one one or more distinct RRVRRV lineage lineage(s)(s) has circulated in PNG and infected inhabitants, in in decades prior to the introductionintroduction ofof thethe PNG PNG clade. clade. Three Three cases cases of RRV-inducedof RRV-induced polyarthritis polyarthritis were documentedwere docu- mentedin the capital, in the capital, Port Moresby Port Moresby between between 1980 and 1980 1981, and potentially 1981, potentially caused caused by strains by relatedstrains relatedto the RRV to the clade RRV described clade described here [13 here]. [13].

4.4. Discussion Discussion OurOur analysis analysis of of the the sole sole RRV RRV isolate isolate to be to derived be derived from from PNG PNG thus thusfar has far shed has shedlight onlight the on molecular the molecular epidemiology epidemiology of this of this medically medically significant signiﬁcant arbovirus arbovirus in in the the greater greater Australasian region. Phylogenetic analysis of the derived genome sequence and 104 available RRV full genome sequences revealed that the PNG isolate constitutes a unique

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genetic group, distinct from all other Australian RRV lineages. The clade is a result of long-term, local evolution of RRV within PNG, independent of Australian variants. The establishment and continuation of RRV transmission in PNG may have been facilitated by one or more of the many endemic marsupial species present in the country [28]. A diversity of mosquito species is present in PNG, many of which have been the source of isolations in field-caught specimens in Australia [16,29]. Differences in vector competence for RRV between species present in Australia and PNG, is not known. An. farauti, the mosquito species from which the PNG isolate was derived, is present in brackish habitats in northern Australia, but has not been associated with RRV transmission in Australia, as of yet. With this new sequence from PNG, the current RRV phylogeny has been expanded. Consistent with our previous analysis of RRV, we estimate that major RRV genetic groups, including the PNG clade, arise approximately every decade. The mean nucleotide substitution rate and tMRCA of the entire RRV dataset, was also consistent with the prior RRV analysis [10]. The mean estimated emergence time of the PNG clade was July 1979, corresponding to peak disease reporting during the PICTs RRV outbreak. The PICTs epidemic was previously thought to have been initiated by an eastern Australian traveler based on the sampling of ‘Eastern’ Australian RRV lineage isolates during the outbreak. The exact origins of the PICTs epidemic viruses, which cluster within G3, are now unclear, as our recent analysis [10] showed G3 viruses were sampled throughout Australia within a 6 year period (1982–1988). RRV disease was not documented in PNG until after the PICTs epidemic, in a 1980 survey of acute arthritis, where 13.6% (of 44 patients) of reported infectious arthritis cases were serologically attributed to RRV infection [14]. Arboviral surveillance, including RRV surveillance, is and has been lacking in PNG with the exact disease epidemiology of RRV within the country, uncharacterised. Serological evidence of RRV was identified in a large proportion of the adult and general population in multiple regions across PNG, in a collection sampled from 1961 to 1969 [11]. This sampling period pre-dates the temporal range estimated obtained in this analysis, by several decades and suggests a separate clade or lineage was in circulation and infecting residents prior to the emergence of the PNG clade. Indeed, if a novel lineage of RRV had been seeded into PNG during the PICTs epidemic, it is feasible that cases went unnoticed, given the potentially high seroprevalence of anti-RRV antibodies in the PNG population. Molecular information of RRV, including the diversity of lineages in past and present circulation, is lacking. Barmah Forest virus is an alphavirus that was thought to be exclusively endemic to Australia, until its recent isolation from a febrile patient in PNG with no history of international travel [30]. Temporal analysis dates the emergence of the unique BFV PNG clade to 50–120 years ago, likely facilitated by movement of large numbers of Australian troops into the Asia Pacific region during World War 2 [31]. Consistent with our findings for RRV, it appears that exchange of BFV between Australia and PNG is sporadic, despite the theoretical potential for more regular spread via mobile vertebrate hosts such as migratory birds [30,31]. Further molecular surveillance of RRV circulating in PNG would be informative to further clarify the molecular epidemiology of RRV in the wider Australasian region, and to characterise the diversity of viruses circulating in the country, and associations to Australian lineages. This analysis has highlighted the long-term circulation of RRV beyond Australia. The potential of RRV to become established in regions beyond the known range of activity, in areas where supportive mosquito vectors and vertebrate hosts are present, should be considered. Indeed, there is need for a collaborative surveillance system between Australia and its regional partners to facilitate on-going, long-term surveillance of RRV and other medically important arboviruses.

Supplementary Materials: The following are available online at https://www.mdpi.com/1999-491 5/13/3/482/s1, Figure S1: Midpoint rooted maximum likelihood phylogeny of a 105-taxon whole- genome Ross River virus dataset, including the sequence of the Papua New Guinea isolate. Figure S2: Viruses 2021, 13, 482 7 of 8

Midpoint rooted maximum likelihood phylogeny of a 105-taxon Ross River virus whole-genome dataset aligned with all publicly available whole or partial E2 gene sequences, that are geographically and temporally defined. Author Contributions: Conceptualization, J.S.M., D.W.S. and A.I.; Data Curation, A.M.; Formal Analysis, A.M.; Funding acquisition, J.S.M., D.W.S. and A.I.; Software, A.I.; Supervision, J.S.M., D.W.S. and A.I.; Writing—Original Draft, A.M. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the Western Australian Department of Health Biological and Applied Environmental Health Hazards Directorate through a PhD scholarship grant funded to support A.M., and by funding provided from PathWest Laboratory Medicine WA and the University of Western Australia. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: The sequence derived in this study was deposited to the NCBI database and assigned the accession number MW238766. Conflicts of Interest: The authors declare no conflict of interest.

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